1. Field of the Invention
The present invention relates generally to a fluorescence detecting device for detecting a fluorescence reaction, for instance, a fluorescence detecting device suitable for detection, etc., of a specific gene contained in a sample.
2. Related Background Art
Recently, the genome sequence analysis has been developed significantly, and the determination of the whole base sequence of the human genome will be completed in 2003. Besides, the determination of genomes of other creatures is proceeding throughout the world. With this development of the genome analysis, the detection of genes has an increased significance from the viewpoint of the determination of functions of genes, the medical diagnosis, etc. Examples of conventional gene detecting methods include the gene amplification methods represented by the polymerase chain reaction (PCR(trademark)) method, while recently the gene detecting method employing DNA chips is used widely.
A DNA chip is an approximately 1 cmxc3x971 cm glass chip, silicon chip, etc. on which a plurality of single-strand DNAs are fixed. Examples of the single-strand DNAs to be fixed include DNAs as etiologic genes. The gene analysis employing a DNA chip is performed, for instance, in the following manner. First of all, a target gene is extracted from cells (for instance, blood cells). Then, the target gene is amplified by the PCR(trademark) method. In the amplification, a fluorescent substance is employed to label an amplification product. A DNA chip is immersed in a solution containing nucleic acid strands labeled with the fluorescent dye, so that hybridization occurs. Thereafter, the DNA chip is washed so that nucleic acids that have not been hybridized are removed.
Subsequently, the DNA chip is irradiated with an excitation light, and the fluorescence is detected. An example of a fluorescence detecting device used herein is shown in FIG. 22. In the device, an excitation light 309 from a light source 305 such as a laser is reflected by a beam splitter 304, and enters an objective lens 306, where the light is focused so as to be incident on a fixed portion 307 of a nucleic acid probe on a DNA chip 308. In the case where a double strand is formed as a result of hybridization, a fluorescent substance is present on the DNA chip 308, and therefore, a fluorescence 310 is emitted upon the irradiation by the excitation light 309. Normally, the fluorescence 310 and the excitation light 309 have a wavelength difference on the order of several tens of nanometers. A part 311 of the fluorescence and a reflected light of the excitation light 309 return to the objective lens, and reach the beam splitter 304. Most of the reflected light of the excitation light 309 is reflected by the beam splitter 304, thereby being directed to the light source side. The part 311 of the fluorescence passes through the beam splitter 304, thereby being directed to a photodetector 301. The part 311 of the fluorescence that has passed through the beam splitter 304 passes through a filter 303 that limits a wavelength, while the reflected light of the excitation light 309 is blocked by the same. Furthermore, the part 311 of the fluorescence passes through a photodetector lens 302 and enters the photodetector 301 for measuring an intensity of the fluorescence, where the fluorescence is detected.
However, the above-described conventional fluorescence detecting device is a large-scale and complex device having a long optical path, through which the fluorescence is lost partly, thereby leading to a problem of low detection sensitivity.
Therefore, with the foregoing in mind, it is an object of the present invention to provide a fluorescence detecting device that is small in size and has a high sensitivity.
To achieve the foregoing object, a first fluorescence detecting device of the present invention includes a semiconductor integrated circuit substrate and a fluorescence reaction vessel where a fluorescence reaction occurs. The semiconductor integrated circuit substrate includes a photodiode and a signal detecting circuit for detecting charges obtained as a result of photoelectric conversion by the photodiode. The fluorescence reaction vessel is arranged above the photodiode. Here, the fluorescence reaction vessel may be displaced from a position immediately above the photodiode as long as at least a part of the fluorescence generated in the fluorescence reaction vessel enters the photodiode. Normally, at least a part of the fluorescence reaction vessel is positioned above the photodiode.
This device is capable of detecting a fluorescence generated as a fluorescence reaction in the fluorescence reaction vessel by the photodiode arranged under the fluorescence reaction vessel. Therefore, an optical path can be shortened, which results in the improvement of the fluorescence detecting sensitivity and the reduction of the overall size of the device.
The first device preferably is configured as follows. The photodiode and the signal detecting circuit compose one unit cell, and a plurality of the unit cells and a circuit for selecting and driving each of the unit cells are formed on the semiconductor integrated circuit substrate. Besides, a plurality of the fluorescence reaction vessels are provided so as to correspond to the unit cells, respectively. The foregoing configuration allows different tests to be performed in the fluorescence reaction vessels, and allows these tests to be performed with one fluorescence measuring operation.
The first device preferably is configured as follows. The photodiode and the signal detecting circuit compose one unit cell, and a plurality of the unit cells and a circuit for selecting and driving each of the unit cells are formed on the semiconductor integrated circuit substrate. Besides, at least one fluorescence reaction vessel is provided so as to be shared by a plurality of the unit cells. This configuration allows a fluorescence of one fluorescence reaction vessel to be detected by a plurality of photodiodes, and hence, such a photodiode is allowed to have a reduced size. Consequently, each photodiode has a smaller capacitance and internal resistance, thereby causing operations such as reading out charges obtained as a result of photoelectric conversion to be performed at a higher speed, which results in high-speed detection of a fluorescence intensity.
In the foregoing preferable configuration, a plurality of the fluorescence reaction vessels preferably are provided so that each of the fluorescence reaction vessels is shared by a plurality of the unit cells. This configuration allows different tests to be performed in the fluorescence reaction vessels, and allows these tests to be performed with one fluorescence measuring operation.
Furthermore, in the first device, a single-strand DNA may be fixed on a bottom of the fluorescence reaction vessel. In this case, it is used as a DNA chip. Alternatively, an antibody or an antigen may be fixed on a bottom of the fluorescence reaction vessel. Furthermore, in the foregoing fluorescence reaction vessel, a gene amplification reaction such as the PCR(trademark) may be carried out so that an amplification product should be detected by fluorescence.
The first device may be produced by, for instance, preparing a transparent substrate in which a cavity or a hole that serves as the fluorescence reaction vessel is formed, and a semiconductor integrated circuit substrate in which the photodiode and the signal detecting circuit are formed, and adhering the transparent substrate and the semiconductor integrated circuit substrate to each other so that the cavity or the hole is positioned above the photodiode. By this producing method, the fluorescence detecting device can be produced readily. This method is effective particularly in the case where a plurality of fluorescence reaction vessels have to be provided.
A fluorescence detecting method employing the first device is a method in which the excitation light is caused to enter the fluorescence reaction vessel, and a fluorescence generated as a result of the entry of the excitation light is detected by means of the photodiode. In this case, the excitation light preferably is caused to enter the fluorescence reaction vessel from a side thereof, so as to prevent the excitation light from entering the photodiode.
To achieve the aforementioned object, a second fluorescence detecting device of the present invention includes a semiconductor integrated circuit substrate and a fluorescence reaction vessel where a fluorescence reaction occurs. The semiconductor integrated circuit substrate includes a photodiode, a charge transfer section for reading out and transferring charges obtained as a result of photoelectric conversion by the photodiode, a charge accumulating section for accumulating the charges transferred thereto by the charge transfer section, and a signal detecting circuit for detecting charged accumulated in the charge accumulating section. The fluorescence reaction vessel is arranged above the photodiode.
This device is capable of detecting a fluorescence generated as a fluorescence reaction in the fluorescence reaction vessel by the photodiode arranged under the fluorescence reaction vessel. Therefore, an optical path can be shortened, which results in the improvement of the fluorescence detecting sensitivity and the reduction of the overall size of the device. Furthermore, in the present device, among the fluorescence incident on the semiconductor integrated circuit substrate side, light corresponding to an aperture ratio of the photodiode enters the same. The aperture ratio of a photodiode significantly increases with the recent development of the micromachining technology, and this also allows the device of the present invention to be capable of detecting a fluorescence with a high sensitivity.
The second device preferably is configured as follows. One unit cell is composed of the photodiode, the charge transfer section, the charge accumulating section, and the signal detecting circuit, and the semiconductor integrated circuit substrate includes a plurality of the unit cells and a circuit for selecting and driving each of the plurality of the unit cells. Besides, a plurality of the fluorescence reaction vessels are provided so as to correspond to the unit cells, respectively. The foregoing configuration allows different tests to be performed in the fluorescence reaction vessels, and allows these tests to be performed with one fluorescence measuring operation.
The second device preferably is configured as follows. The semiconductor integrated circuit substrate includes a plurality of the photodiodes, the fluorescence reaction vessel is arranged so that a plurality of the photodiodes treats a fluorescence from the fluorescence reaction vessel, and charges obtained as a result of photoelectric conversion by the plurality of the photodiodes are summed by at least one of the charge transfer section and the charge accumulating section. This configuration causes an increased quantity of signal charges to be detected by the signal detecting circuit, thereby enabling the detection with a further improved sensitivity.
In this case, a plurality of the fluorescence reaction vessels may be provided. With the provision of a plurality of fluorescence reaction vessels, if the signal detecting circuit is provided with respect to each fluorescence reaction vessel, variations between the circuits could adversely affect the detection of the signals of the plurality of the fluorescence reaction vessels. Therefore, a singular signal detecting circuit preferably is provided.
Furthermore, in the second device, a single-strand DNA may be fixed on a bottom of the fluorescence reaction vessel. In this case, it is used as a DNA chip. Alternatively, an antibody or an antigen may be fixed on a bottom of the fluorescence reaction vessel. Furthermore, in the foregoing fluorescence reaction vessel, a gene amplification reaction such as the PCR(trademark) may be carried out so that an amplification product should be detected by fluorescence.
The second device can be produced by, for instance, preparing a transparent substrate in which a cavity or a hole that serves as the fluorescence reaction vessel is formed, and a semiconductor integrated circuit substrate in which the photodiode, the charge transfer section, the charge accumulating section, and the signal detecting circuit are formed, and adhering the transparent substrate and the semiconductor integrated circuit substrate to each other so that the cavity or the hole is positioned above the photodiode. By this producing method, the fluorescence detecting device can be produced readily. This method is effective particularly in the case where a plurality of fluorescence reaction vessels have to be provided.
A fluorescence detecting method employing the first device is a method in which the excitation light is caused to enter the fluorescence reaction vessel, and a fluorescence generated as a result of the entry of the excitation light is detected by means of the photodiode. In this case, the excitation light preferably is caused to enter the fluorescence reaction vessel from a side thereof, so as to prevent the excitation light from entering the photodiode.